Antifungal activity of the essential oil of Thymus capitellatus against Candida, Aspergillus and dermatophyte strains

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1 FLAVOUR AND FRAGRANCE JOURNAL Flavour Fragr. J. 2006; 21: Published online 24 May 2006 in Wiley InterScience ( ANTIFUNGAL ACTIVITY OF THYMUS CAPITELLATUS 749 Antifungal activity of the essential oil of Thymus capitellatus against Candida, Aspergillus and dermatophyte strains Lígia Ribeiro Salgueiro, 1 * Eugénia Pinto, 2 Maria José Gonçalves, 1 Inês Costa, 1 Ana Palmeira, 2 Carlos Cavaleiro, 1 Cidália Pina-Vaz, 3 Acácio Gonçalves Rodrigues 3 and José Martinez-de-Oliveira 4 1 Laboratório de Farmacognosia, Faculdade de Farmácia/CEF, Universidade de Coimbra, 3000 Coimbra, Portugal 2 Serviço de Microbiologia/CEQOFF, Faculdade de Farmácia, Universidade do Porto, 4050 Porto, Portugal 3 Serviço de Microbiologia, Faculdade de Medicina, Universidade do Porto, 4200 Porto, Portugal 4 Departamento de Ginecologia e Obstetrícia, Faculdade de Medicina, Universidade do Porto, 4200 Porto, Portugal Received 15 November 2004; Revised 28 January 2005; Accepted 7 February 2005 ABSTRACT: The antifungal activity of Thymus capitellatus oils on Candida, Aspergillus and dermatophyte strains were studied. The essential oils were obtained from the aerial parts of the plants by water distillation and analysed by GC and GC MS. Three chemotypes were characterized: 1,8-cineole (47.5%), 1,8-cineole/borneol (28.8% and 19.5%, respectively) and 1,8-cineole/linalyl acetate/linalool (27.5%, 20.0% and 17.0%, respectively). The minimal inhibitory concentration (MIC) determined according to the NCCLS protocols (M27-A and M38-P) and the minimal lethal concentration (MLC) were used to evaluate the antifungal activity of the oils against Candida (seven clinical isolates and three ATCC type strains), Aspergillus (five clinical isolates, two CECT and two ATCC type strains) and five dermatophyte clinical fungi strains. The oils exhibited antifungal activity for the dermatophyte strains, with MIC values of µl/ml; the chemotype 1,8-cineole/linalyl acetate/linalool proved to be more active. The highest antifungal activity of this oil can be associated with the contribution of the linalyl acetate. In the other hand, all samples showed low activity against Candida and Aspergillus strains. Copyright 2006 John Wiley & Sons, Ltd. KEY WORDS: Thymus capitellatus; Lamiaceae; essential oil composition; antifungal activity Introduction Thymus capitellatus Hoffmanns. & Link (family Lamiaceae; local name tomilho do mato ) is an endemic aromatic plant from Portugal 1 which grows in the estuaries and down-river parts of the Tejo and Sado basins (Estremadura, Ribatejo and Alentejo provinces). Studies on the essential oils obtained from collective 2,3 and individual samples 4 of T. capitellatus demonstrated that this species is polymorphic, with three main chemotypes (1,8-cineole; 1,8-cineole/borneol and 1,8- cineole/linalyl acetate/linalool chemotypes). Our previous results showed that the 1,8-cineole and 1,8-cineole/ borneol chemotypes are well distributed in the provinces of Ribatejo and Alentejo, whereas the 1,8-cineole/linalyl acetate/linalool chemotype is widespread only in the province of Estremadura. We verified that in Estremadura province other Thymus species, such as T. carnosus and * Correspondence to: L. R. Salgueiro, Lab. de Farmacognosia, Fac. de Farmácia/CEF, Universidade de Coimbra, 3000 Coimbra, Portugal. ligia@ff.uc.pt Contract/grant sponsor: FCT POCTI, Portugal; contract/grant number: POCTI/40167/ESP/2001. Contract/grant sponsor: FEDER, Portugal. T. mastichina, were also characterized by high linalool content, whereas other samples coming from various regions of Portugal have very low amounts of this compound. 5,6 This coast has a high Atlantic maritime humidity concentration, which seems to be correlated with the amount of linalool. In some localities of the Estremadura, T. capitellatus is regarded as an antiseptic and is usually used in the treatment of cutaneous infections. The objective of the present research is to determine the antifungal activity of chemically well-defined chemotypes of T. capitellatus on Candida, Aspergillus and dermatophyte fungal strains. To our knowledge, this is the first report on the antifungal activity of the essential oil of this species. Material and Methods Plant Material Aerial parts of the plants were collected at the flowering stage from two different localities of Ribatejo (Porto Alto, sample A, and Samora Correia, sample C) and one locality of Estremadura (Poceirão, sample B). Voucher

2 750 L. R. SALGUEIRO ET AL. specimens were deposited at the Herbarium of the Instituto Batânico of the University of Coimbra (COI), under Accession Nos LS Essential Oil Analysis The essential oil content of the air-dried plant material was determined according to the European Pharmacopoeia method. 7 Analysis of volatile oils obtained by water distillation for 3 h were carried out by GC and GC MS, using fused silica capillary columns with two different stationary phases: SPB-1 (polydimethylsiloxane 30 m 0.20 mm i.d., film thickness 0.20 µm), and Supelco Wax 10 (polyethyleneglycol 30 m 0.20 mm i.d., film thickness 0.20 µm); oven temperature programme, 70 C to 220 C at 3 C/min, then held at 220 C for 15 min; injector temperature, 250 C; detector carrier gas, helium, adjusted to a linear velocity of 30 m/s; split ratio, 1:40; temperatures, 250 C. GC MS was performed with a HP1 fused silica column (polydimethylsiloxane 30 m 0.25 mm i.d., film thickness 0.25 µm), interfaced with a mass selective detector. GC parameters were as above; interface temperature, 250 C; MS source temperature, 230 C; MS quadrupole temperature, 150 C; ionization energy, 70 ev; ionization current, 60 µa; scan range, u; scans/s, The identity of the components was achieved from their retention indices, calculated by linear interpolation relative to retention times of a series of n-alkanes, and their mass spectra, which were compared with those from our own library and literature data. 8,9 Relative amounts of individual components were calculated based on GC peak areas without FID response factor correction. Antifungal Evaluation Antifungal activity of the three chemotypes was evaluated against Candida, Aspergillus and dermatophyte strains: seven Candida clinical strains, two of C. albicans (M1, H37), one of C. krusei (H9), one of C. tropicalis (H18), one of C. guillermondii (Mat23) and two of C. glabrata (H16, H30) isolated from recurrent cases of vulvovaginal candidosis, as well as three ATCC type strains (C. albicans ATCC 10231, C. tropicalis ATCC and C. parapsilosis ATCC 90018); five Aspergillus clinical strains, one of A. niger (F01), three of A. fumigatus (F05, F07, F17) and one of A. flavus (F44) isolated from bronchial secretions, as well as two ATCC type strains (A. niger ATCC and A. fumigatus ATCC 46645) and two CECT type strains (A. niger CECT 2574 and A. fumigatus CECT 2071); five dermatophyte clinical strains (Microsporum canis FF1, M. gypseum FF3, Trichophyton rubrum FF5, T. mentagrophytes FF7 and Epidermophyton floccosum FF9) isolated from nails and skin. MICs and MLCs were determined by a macrodilution method according the NCCLS protocols (M27-A and M38-P). 10,11 Antifungal activity of the major constituents of each chemotype (1,8-cineole, borneol, linalyl acetate and linalool) were also evaluated against dermatophyte strains. Fluconazole (Sigma) and amphotericin B (Pfizer) were used to control the sensitivity of the tested microorganisms. The two-fold serial dilutions that were used varied in the range µg/ ml for fluconazole, µg/ml for amphotericin B and µl/ml (v/v) for the essential oils and their major components. Three independent experiments performed in duplicate were realized for each chemotype (A, B and C). Results and Discussion The oils were obtained in yields of % (v/w). The qualitative and quantitative compositions of the three chemotypes (A, B and C) used for antifungal evaluation are shown in Table 1, where the compounds are listed in order of their elution on a polydimethylsiloxane column. In total, 67 compounds were identified, accounting for % of the essential oils. The three chemotypes were characterized by a high percentage of oxygenated monoterpenes ( %) and monoterpene hydrocarbons ( %). Sesquiterpenes attained %. All three oils had a high 1,8-cineole content ( %). Nevertheless, there were some important differences between them. In chemotype A the 1,8-cineole was the major component (47.5%), whereas in the chemotypes B and C there were other main compounds: in chemotype B linalyl acetate (20.0%) and linalool (17.0%) and in chemotype C borneol. Evaluation of minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of the three chemotypes of T. capitellatus showed antifungal activity against dermatophyte strains, with MIC and MLC values of µl/ml (Table 2). Nevertheless, the 1,8- cineole/linalyl acetate/linalool chemotype (B) proved to be more active. Antifungal activity of the major constituents of the three chemotypes (1,8-cineole, borneol, linalyl acetate and linalool) were also assayed against the same dermatophytes. The highest antifungal activity of chemotype B can be associated with the contribution of the linalyl acetate (Table 3). These results support the popularity of this plant in the treatment of cutaneous infections and may justify the future use of T. capitellatus oils, particularly these with high amounts of linalyl acetate, in the treatment of dermatophytosis. For Candida and Aspergillus strains the three oils showed low activity, with MIC values of µl/ml (Table 2). C. tropicalis and A. flavus showed higher resistance to the oils.

3 ANTIFUNGAL ACTIVITY OF THYMUS CAPITELLATUS 751 Table 1. Percentage composition of three chemotypes of T. capitellatus from Portugal Compound RI a RI b Content (%) A B C Tricyclene α-thujene α-pinene Camphene Sabinene β-pinene Myrcene α-phellandrene t t α-terpinene p-cymene Limonene ,8-Cineole (Z)-β-Ocimene (E)-β-Ocimene γ -Terpinene trans-sabinene hydrate t 0.6 cis-linalool oxide Fenchone t t 0.1 trans-linalool oxyde t cis-sabinene hydrate Terpinolene Linalool α-campholenal t 0.1 Camphor trans-pinocarveol trans-verbenol Pinocarvone t 0.2 δ-terpineol Borneol p-cimene-8-ol t Terpinene-4-ol Myrtenal t 0.3 α-terpineol Verbenone Myrtenol t 0.2 trans-carveol t Nerol Carvone t t 0.2 Neral t Geraniol t Linalyl acetate t 20.0 t Bornyl acetate α-terpenyl acetate Neryl acetate t 0.7 t α-cubebene t t t Geranyl acetate t 1.5 t β-bourbonene t β-cubebene t t β-elemene α-gurjunene (E)-Caryophyllene α-humulene allo-aromadendrene Germacrene D Bicyclogermacrene t t β-bisabolene t 0.1 γ -Cadinene δ-cadinene t t Elemol Caryophyllene oxide Viridiflorol Ledol epi-γ-Eudesmol Cubenol T-Cadinol α-cadinol Intermedeol 1630 n.d

4 752 L. R. SALGUEIRO ET AL. Table 1. (Continued) Compound RI a RI b Content (%) A B C Monoterpene hydrocarbons Oxygen-containing monoterpenes Sesquiterpene hydrocarbons Oxygen-containing sesquiterpenes Total identified Compounds listed in order to their elution on the SPB-1 column. t, trace (<0.05%). RI a, Retention indices on the SPB-1 column relative to C 8 C 22 n-alkanes. RI b, Retention indices on the SupelcoWax-10 column relative to C 8 C 22 n-alkanes. n.d., not determined. Table 2. Antifungal activity (MIC and MLC) of the three chemotypes of T. capitellatus for Candida, dermatophyte and Aspergillus strains Strains Chemotype A Chemotype B Chemotype C Fluconazole Amphotericin B MIC a MLC a MIC MLC MIC MLC MIC b MLC b MIC MLC Candida albicans ATCC >128 N.T c N.T C. albicans H >128 N.T N.T C. albicans M N.T N.T C. tropicalis ATCC >128 N.T N.T C. tropicalis H >128 N.T N.T C. glabrata H N.T N.T C. glabrata H N.T N.T C. krusei H N.T N.T C. guillermondii MAT N.T N.T C. parapsilosis ATCC <1 <1 N.T N.T Epidermophyton floccosum N.T N.T Trichophyton rubrum N.T N.T T. mentagrophytes N.T N.T Microsporum canis N.T N.T M. gypseum N.T N.T Aspergillus niger ATCC N.T N.T A. niger CECT N.T N.T 2 4 A. niger F N.T N.T 1 2 A. fumigatus ATCC N.T N.T 2 4 A. fumigatus CECT N.T N.T A. fumigatus F N.T N.T A. fumigatus F N.T N.T A. fumigatus F N.T N.T A. flavus F N.T N.T 2 8 a MIC and MLC were determined by a macrodilution method and expressed in µl/ml (v/v). b MIC and MLC were determined by a macrodilution method and expressed in µg/ml (w/v). c Not tested. Table 3. Antifungal activity (MIC and MLC) of the major compounds of the essential oil of T. capitellatus for dermatophyte strains Strains 1,8-Cineole Borneol Linalool Linalyl acetate MIC (a) MLC (a) MIC MLC MIC MLC MIC MLC Epidermophyton floccosum Trichophyton rubrum T. mentagrophytes Microsporum canis M. gypseum a MIC and MLC were determined by a macrodilution method and expressed in µl/ml (v/v). b MIC and MLC were determined by a macrodilution method and expressed in µg/ml (w/v).

5 ANTIFUNGAL ACTIVITY OF THYMUS CAPITELLATUS 753 Acknowledgements This study was supported by FCT POCTI and FEDER (POCTI/40167/ESP/2001) and CEF (University of Coimbra). References 1. Franco JA. Nova Flora de Portugal (Continente e Açores), vol II. Franco A, Lisboa, 1984; Velasco Negueruela A, Perez Alonso MJ, Burzaco A. Aceites esenciales de tomillos ibéricos. IV. Contribución al conocimiento del aceite esencial de Thymus capitellatus. An Jard. Bot. Madrid 1991; 49: Figueiredo AC, Barroso JG, Pais MS, Scheffer JJC. The essential oils of two endemic Portuguese thyme species: Thymus capitellatus and T. lotocephalus. Flavour Fragr. J. 1993; 8: Salgueiro LR. Os tomilhos portugueses e os seus óleos essenciais. Doctoral thesis, University of Coimbra, 1994; Salgueiro LR, Vila R, Tomás X et al. Chemical polymorphism of the essential oil of Thymus carnosus from Portugal. Phytochemstry 1995; 38: Salgueiro LR, Vila R, Tomás X et al. Composition and variability of the essential oils of Thymus species from section Mastichina from Portugal. Biochem. Syst. Ecol. 1997; 7: Council of Europe. European Pharmacopoeia, 3rd edn. Council of Europe: Strasbourg, 1997; Adams RP. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured: Carol Stream, IL: Joulain D, König WA. The Atlas of Spectral Data of Sesquiterpene Hydrocarbons. E.B.-Verlag: Hamburg, National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved Standard vol 27. M27-A, National Committee for Clinical Laboratory Standards: Wayne, PA, 1997; National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of conidium-forming filamentous fungi. Proposed standard M38-P, vol 18. National Committee for Clinical Laboratory Standards: Wayne, PA, 1998; 13.